Rous-associated virus-2 (RAV-2) is an avian leukosis virus initially isolated from stocks of Rous sarcoma virus (RSV). (7). When injected into one-day-old ...
JOURNAL
OF
VIROLOGY, Feb. 1984. p. 557-565
Vol. 49. No. 2
0022-538X/84/02)0557-09$02.00/0 Copyright C 1984. American Society for Microbiology
Nucleotide Sequence of Noncoding Regions in Rous-Associated Virus-2: Comparisons Delineate Conserved Regions Important in Replication and Oncogenesis DIANE BIZUB, RICHARD A. KATZ. AND ANNA MARIE SKALKA* Roche Instituite of Molecliar Biology, Roche Reseanch Ceniter-, Ntiley, New Jersey 07110 Received 15 August 1983/Accepted 18 October 1983
The nucleotide sequence of the regions flanking the long terminal repeat of Rous-associated virus-2 has been determined. The region analyzed spans the ends of the viral genome and includes the terminus of the enm' gene, the 3' noncoding region. the 5' noncoding region. and the beginning of the gag gene. These data have been compared with sequences available from other avian retroviruses. The comparisons reveal sections which are highly conserved and others which are quite variable. Sequence homologies within the conserved regions suggest details concerning the mode of origin of the si-c-transducing viruses. Included in the variable section is a region (XSR) found only in certain strains of Rous-derived virus. Its absence from other oncogenic viruses indicates that these sequences are not required to elicit disease.
Rous-associated virus-2 (RAV-2) is an avian leukosis virus initially isolated from stocks of Rous sarcoma virus (RSV) (7). When injected into one-day-old chickens, RAV-2 and other avian leukosis viruses cause a high incidence of bursal lymphomas after a period of 4 to 12 months. It is now known that these retroviruses, which lack an oncogene, induce such tumors by activating c-mnv, the cellular homolog of the oncogene of MC29 virus (8, 20). This activation is presumed to depend on the transcription promoter or enhancer function encoded in the long terminal repeat (LTR) of a provirus which integrates in close proximity to c-mvc. These proviruses are almost always defective; some contribute little more than an LTR at the c-mvc locus (19, 20). In a recent analysis of retroviral nucleotide sequences presumed to be required for oncogenicity, Tsichlis et al. (29) compared data obtained from a weakly oncogenic recombinant virus, NTRE-7, and its oncogenic exogenous and nononcogenic endogenous parents. These analyses showed that NTRE-7 inherited a segment which included the U3 region of the LTR and an adjacent region of ca. 140 base pairs (called XSR) from the exogenous parent, the Prague strain of RSV (PR-RSV). It seemed most likely that the element responsible for oncogenicity in this case, as in the cmvc activation described above, was the promoter-enhancer function encoded in the U3 region of the exogenous viral LTR. However, a contribution from the 3' XSR sequences could not be excluded at that time. Our laboratory has reported the nucleotide sequence of the RAV-2 LTR (14). In the present study we extended these analyses to include the 3' and 5' viral noncoding regions to determine if RAV-2 contains sequences similar to the XSR of NTRE-7. In our comparisons, we surveyed sequences from analogous regions of other retroviruses. The results show that the XSR sequence is not present in RAV-2 or several other oncogenic viral genomes studied, and thus it does not appear to be essential for oncogenicity. Our data also reveal homologies which suggest that the .src oncogene could have been captured by RSV via a mechanism which included homologous recombination.
*
MATERIALS AND METHODS Clones. All clones used for sequencing were derived from X RAV2-2. As previously reported (13), A RAV2-2 was generated by inserting HindIII-digested RAV-2 covalently closed circular DNA containing one or two copies of the LTR into the A cloning vector Charon 21A. Further subcloning involved insertion of the appropriate fragment from Sail and BalrHI digested A RAV2-2 DNA into plasmid (pBR322) and other phage (M133mp9) vectors. Two other RAV-2derived clones which were utilized, mp2-R2.1(-) and mp2R2.1(+), contain an EcoRi insert equivalent to a single LTR in either orientation (14). DNA sequencing. The sequencing strategy is illustrated in Fig. 1. Sequencing by chemical cleavage was performed on both strands of the Sal-Barn pBR322 clone (pGJ14, provided by Grace Ju) as described by Maxam and Gilbert (18). starting about 320 base pairs away from the Sall site. The dideoxy chain termination method was used with both the mp2 and mp9 M13 subclones (9, 21). Exonuclease III sequencing was done as described by Guo and Wu (6) by using replicative form I DNA from the mp9 subclone cut with Splil. RESULTS AND DISCUSSION The region included in our sequence analysis of RAV-2 is indicated in the map in Fig. 1. We used recombinant DNA clones which originated from intracellular covalently closed circular viral DNA containing one copy of the LTR. The derived sequence (Fig. 2) runs from the end of the eni' gene through the adjacent noncoding region. the LTR. and another noncoding region which corresponds to the 5' end of the virus. The analyses end in the sequences which encode the amino terminus of p19 in the viral gag gene. Table 1 lists the viral sequences we have compared and the sections included in subsequent figures and in the summary diagram (see Fig. 9). The nucleotide and predicted amino acid sequence at the end of the env gene of RAV-2 and other retroviruses. Data from RAV-2 was compared with information available for two other Rous-derived viruses: a subgroup A SchmidtRuppin strain (SR-RSV) and, subgroup C. PR-RSV. Where relevant, data from another sarcoma virus, Y73, avian myeloblastosis virus (AMV), and the nononcogenic sub-
Corresponding author. 557
J. VIROL.
BIZUB, KATZ, AND SKALKA
558
s,al1I
2.00
Maxom-Gilbert Sequencing
03
_+
~ *
776 700 900
Barn f->gag1
R 05
02~1 II22 1043
300 1200
1 101 40C
.*-
.
4-
Dideoxy Sequencing Exo M Sequencing
1560
FIG. 1. Sequencing strategy. The figure shows a map of the ends of RAV-2 joined at the LTR as it occurs in clones derived from intracellular covalently closed circular viral DNA molecules. The heavy line shows the region sequenced, numbered as indicated from left to right. Results from three sequencing methods were combined (see the text). Arrows indicate the direction and origin of sections analyzed. DIRECT
REPEATHl
PPT
JrU3
AACTTGACAACATCACTCCTCGGGGACTTATTAGATGATGTCACGAGTAT 50
GTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACTCTT r TR 01
TCGACACGCAGTCCTGCAGAACCGAGCGGCTATTGACTTCTTGCTCCTAG 100
GTAGTCTTGCAACATGCTTATGT^ACGATGAGTTAGCAACATGCCTTATA 850
CTCACGGCCATGGCTGTGAGGACATTGCCGGAATGTGTTGTTTCAATCTG 150
AGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGGAGTAAGGTGGTATG 900
AGTGATCACAGTGAGTCTATACAGAAGAAGTTCCAGCTAATGAAGGAACA 200
ATCGTGGTATGATCGTGCCTTGTTAGGAAGGCAACAGACGGGTCTAACAC 950
TGTCAATAAGATCGGCGTGAACAACGACCCAATCGGAAGTTGGCTGCGAG 250
GGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGT 1000
GATTATTCGGAGGAATAGGAGAATGGGCCGTACACTTGCTGAAAGGACTG 300
[U5 U3J1 REPEAT GCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTG
800
CAP
1050
CTTTTGGGGCTTGTAGTTATCTTGTTGCTAGTAGTATGCTTGCCTTGCCT 350 +1/
TTTGCAATGTGTATCTAGTAGTATTCGAAAGATGATTGATAATTCACTCG 400 GCTATCGCGAGGAATATAAAAAAATTACAGGAGGCTTATAAGCAGCCCGA 450
(ENV]
CACCTGGGTTGATGG C/T CGGACCGTTGATTCCCTGACGACTACGAGC 1096 +1U5]r PBS ] AC C/A TGCATGAAGCAGAAGGCTTCATTTGGTGACCCCGACGTGATCG 1142
r
IR
l
I
A A PA A rArPrT A orrr A rT'rTYrs ATTrrrTrTrA TArrTrrTTnrA TT CAn UMbL 11A/1 IbUO AAGAAGGbbLbIAbGALbTbi ICTIbIlI IbIbITTTA
TTAGGGAATAGTGGTCGGCCACAGGCGGCGTGGCGATCCTGTCCTCATCC
1192
GGTAATTGATCGGCTGGCACGCGGAATATAGGAGGTCGCTGAATAGTAAA 550
GTCTCGCTTATTCGGGGAGCGGACGATGACCCTAGTAGAGGGGGCTGCGG
1242
CTTGTAGACTTGGCTACAGCATAGAGTATCTTCTGTAGCTCTGATGACTG
600
CTTAGGAGGGCAGAAGCTGAGTGGCGTCGGAGGGAGCCCTACTGCAGGGG
1292
CTAGGAAATAATGCTACGGATAATGTGGGGAGGGCAAGGCTTGCGAATCG 650
GCCAACATACCCTACCGAGAACTCAGAGAGTCGTTGGAAGACGGGAAGGA
1342
[DIRECT REPEAT GGTTGTAACGGGCAAGGCTTGACTGAGGGGACAATAGCATGTTTAGGCGA 700
AGCCCGACGACTGAGCGGTCCACCCCAGGCGTGATTCCGGTTGCTCTGCG 1392
AAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTA 750
TGATTCCGGTCGCCCGGTGGATCAAGCATGGAAGCCGTCATAAAGGTGAT 1442 fFGAG> TTCGTCCGCGTGTAAGACCTATTGCGGGAAAACCTCTCCTTCTAAGAAGG 149?
AAATAGGGGCTATGTTGTCCCTGTTACAAAAGGAAGGGTTGCTTACGTCC 1542 CCCTCAGACTTATATTCC
1560
FIG. 2. Nucleotide sequence of the ends of the RAV-2 genome. The sequence begins 152 base pairs downstream of the start of the gp37 section in the envelope gene (eni'). Ent' ends at nucleotide 438. The region from enm' to the start of the LTR includes a section homologous to one first identified as a direct repeat (DR) on either side of src in SR-RSV (3). Its limits (nucleotides 654 to 765) are indicated. A polypurine tract (PPT) (nucleotides 766 to 776) lies between the DR section and the beginning of the LTR. The U3 region of the LTR begins at nucleotide 777 and ends at nucleotide 1021. Between the end of U3 and the beginning of U5 is a 21-nucleotide sequence which is repeated at either end of the viral RNA genome (R). The U5 region begins at nucleotide 1043 and ends at nucleotide 1122. The ambiguities at positions 1066 and 1099 indicate differences obtained in analysis of two M13 clones which were derived from the same fragment inserted in opposite orientations (14). At position 1066, the (+) orientation showed a C, whereas the (-) showed a T; at position 1099 the (+) orientation was a C, whereas the (-) was an A. These differences were confirmed, and we presume they were the result of random mutation within the insert. An IR region in the LTR is located between nucleotides 777 and 791 and between nucleotides 1108 and 1122. The cap site for viral mRNA is located at position 1022, and the tRNA"'P primer binding site (PBS) is located from positions 1123 to 1140. The gag gene begins at position 1420.
NUCLEOTIDE SEQUENCE OF NONCODING REGIONS IN RAV-2
VOL. 49, 1984
RAV-2 PR-RSV( 1) PR-RSV(2) SR-RSV(1)
AACTTGACAACATCACTCCTCGGGGACTTATTAGATGATGTCACGAGTAT 50 --------------------------------G----------------- 6471 ______ _________________-------- G----------------_______ ____________ _ ___------ G----------------_ _____ _________ _ __---------- G-----------------
RAV-2 Y73 PR-RSV(1) PR-RSV(2) SR-RSV(1) SR-RSV(2)
TCGACACGCAGTCCTGCAGAACCGAGCGGCTATTGACTTCTTGCTCCTAG 100
RAV-2 Y73 PR-RSV(1)
CTCACGGCCATGGCTGTGAGGACATTGCCGGAATGTGTTGTTTCAATCTG 150 ----T-------------------------------------------------------------------G-------------------------- 6571 ----------------------- G-------------------------------------------------G----T-------- C---------T--
SR-RSV(2)
PR-RSV(2) SR-RSV(1) SR-RSV(2)
---------G-----------------------------------T---- 6521
-----G--------- -------- ------------------ T------------G--------------------------T------------G------------------G------------____ __________
------------------------G-------------C---------T--
Y73
AGTGATCACAGTGAGTCTATACAGAAGAAGTTCCAGCTAATGAAGGAACA 200 -------------------------T------------------------------------------------------------------A---- 6621
SR-RSV(2)
--------
RAV-2 Y73 PR-RSV( 1) PR-RSV(2) SR-RSV( 1) SR-RSV(2)
TGTCAATAAGATCGGCGTGAACAACGACCCAATCGGAAGTTGGCTGCGAG 250 C-----------T------G---G--------------------------
RAV-2 Y73
GATTATTCGGAGGAATAGGAGAATGGGCCGTACACTTGCTGAAAGGACTG 300
RAV-2
PR-RSV( 1) PR-RSV(2) SR-RSV( 1)
G-----------------------------------------
-------------------G---G-------------------------- 6671 -------------------G---G--------------------------------------------G---G---------T---------------- ------------------G-T-G ----T---T--------------_
-------------------------------T--T---------------
PR-RSV( 1) PR-RSV(2) SR-RSV(1) SR-RSV(2)
-GA-------G--------G-----------T--TC----A--------- 6721 -G -------- G-------- G -----------T--TC ----A---------
RAV-2 PR-RSV( 1) PR-RSV(2) SR-RSV(1 ) SR-RSV(2)
CTTTTGGGGCTTGTAGTTATCTTGTTGCTAGTAGTATGCTTGCCTTGCCT 350
RAV-0
---------------------T--------------G---C-------------------G---C----------
RAV-2
TTTGCAATGTGTATCTAGTAGTATTCGAAAGATGATTGATAATTCACTCG 400
--C ------- G--------------------T--T ----------------C ------G--------------------T--T---------------
--------------------------A--------G--TC----------
Y73
Y73
PR-RSVY(1) PR-RSV (2) SR-RSV(1) SR-RSV(2) RAV-0 RAV-2 Y73 PR-RSV( 1) PR-RSV(2) AMV SR-RSV(1) SR-RSV(2)
RAV-0
--------------------T--A------C-G--G---C---------- 6771 -T--A-- -------- ---G- --C-- ----- ---
- ---- ---- -- -- -- - - --
--------------------T--A------------G---C----------
--- ----GT-------- C--C--C--G --------- A-C--C ---T--A ---A----T---G------------------------A---G----A--A 6821 --A - ----T ----G- -- -- -- -- -- -- -- -- -- -- ----A----G- ---A --A ------ATC ----GCG---AC--CA ----------- A----C--CA--A ------ATGT---GCG---A--GGA ----------- A----C--CA--A ------AT ---G--C -----C--C ------------A--------A--A -
